This proposal is a renewal of our previous project establishing X. tropicalis as a new model for vertebrate developmental genetics. We have made substantial progress on several fronts: 1) animal husbandry is significantly improved; 2) over 40 transgenic lines have been established and used to help resolve several important embryological questions; 3) inbred lines have been established, and polymorphisms are beginning to be mapped; 4) a conditional gene expression system has been developed; 5) chemical and insertional mutagenesis strategies are underway; and 6) a number of developmental mutants have been isolated and are being characterized. We now propose another round of improvements in animal husbandry to further shorten generation time. We will also use our conditional gene expression system to develop new ways to study genes whose function is essential for formation of particular tissues in the embryo. The main thrust of this renewal application is to undertake screens for new mutations, using chemical and insertional mutagenesis strategies for identifying recessive phenotypes. In addition, we describe a strategy for identifying gain-of- function phenotypes. A number of approaches will be developed for characterizing newly identified mutations, including plans for identification of the developmental processes and gene defects responsible for them. Finally, preliminary experiments are described to lay the groundwork for a gene targeting strategy in X. tropicalis. This project should complement the growing genomic resources for X. tropicalis which include a genome project to commence in the summer of 2002.